Beta cells of the mature functional pancreas regulate metabolic homeostasis by controlled secretion of insulin. Impaired beta cell function and the resulting insufficient secretion of insulin, lead to persistently elevated levels of blood glucose, the hallmark of diabetes.
Type 1 diabetes (T1D, also known as insulin dependent diabetes mellitus, IDDM) is an autoimmune disease that results in the destruction of the beta-cells in the pancreas. The collapse of glucose homeostasis and clinical manifestation of the disease is thought to occur only after 80-90% of pancreatic beta cells have been inactivated by the immune response. Incipient diabetes can be diagnosed by the detection of immunological markers of beta cell autoimmunity only after the onset of the autoimmune process.
Type 2 Diabetes (T2D, formerly non-insulin-dependent diabetes mellitus, NIDDM, or adult-onset diabetes) is the most common form of diabetes, accounting for 90% of cases of diabetes. It is a metabolic disorder that is characterized by high blood glucose in the context of relative insulin resistance and insulin deficiency. Obesity is the primary cause of T2D in people who are genetically predisposed to the disease. Type 2 diabetes is initially managed by increasing exercise and dietary modification. If blood sugars are not lowered by these measures, medications such as metformin or insulin may be needed.
Current treatments for diabetes are problematic and do not eliminate many of the long term complications associated with the disease. Consequently, there is great interest in developing improved treatments for diabetes.
The adult pancreas is composed of endocrine and exocrine cell populations. The endocrine cells are located within the islets of Langerhans, comprising cell-types of discrete functionalities (e.g. alpha cells producing glucagon, beta cells producing insulin, delta cells producing somatostatin etc.). In addition to the heterogeneity of the islets themselves, islet preparations are often contaminated by varying fractions of exocrine cells (including acinar cells, characterized by hydrolytic enzymes e.g. trypsin, chymotrypsin, amylase, lipase; and duct cells (Cleveland et al. 2012)) and even non-pancreatic cells. Pancreatic beta cells regulate metabolic homeostasis by controlled secretion of insulin; impaired beta cell function leads to persistently elevated levels of blood glucose, the hallmark of diabetes. Transplantation of functional pancreatic beta cells is one of the most promising approaches towards curing diabetes (Shapiro et al. 2000, N Engl J Med 343(4): 230-238; Serup et al. 2001, Bmj 322(7277): 29-32), but is currently limited by a severe shortage of donor tissue. This has motivated approaches capable of in vitro generation of functional insulin-producing cells (Kroon et al. 2008, Nat Biotechnol 26(4): 443-452; Russ et al. 2011, PLoS One 6(9): e25566). However, the lack of cell type-specific surface markers is a major obstacle for isolation of relevant cells.
The ongoing world-wide epidemic of diabetes emphasizes the urgent need for improved diabetes treatments such as cell based therapies, hence the enormous interest in hESCs-derived pancreatic precursors. Recent results have demonstrated the potential of hESC-derived precursors to produce insulin in vivo (Kroon et al., Nat Biotechnol. 2008, 26,4, 443-52); yet the protocols are very inefficient and yield highly heterogeneous populations. Therefore, any future clinical use would likely require prospective identification and generation of precursor populations with higher fidelity.
One approach is to generate beta cells in vitro from human embryonic stem cells (hESCs). hESCs are pluripotent cells, capable of generating every cell of the body. Since their initial derivation, hopes have been high that these cells might represent a source of unlimited numbers of beta cells, or beta cell progenitors for transplantation to diabetics. Another potential source is based on in vitro expansion of adult beta cells. Such expansion is accompanied by loss of beta cell markers and requires re-differentiation procedures to restore insulin expression (Russ et al., Diabetes. 2008, 57, 1575-83; Russ et al., PLoS One. 2011, 6, 9, e25566). Another serious impediment to cell replacement therapy is the current lack of cell surface markers selective for pancreatic cell subtypes.
Current methods for identification of cell surface markers are based on flow cytometry, analysis of gene expression patterns and immunostaining. Higher throughput proteomics approaches based on antibody arrays, have also been used for profiling cell surface markers (Ko et al. 2005, Biomaterials 26(23): 4882-4891) and for discriminating cell populations based on differentially expressed markers (Sharivkin et al. Molecular & cellular proteomics 2012, 11(9): 586-595; and Belov et al. Cancer Res. 2001, 61, 4483-4489). Sharivkin et al. and Belov et al. have used antibody array procedures to screen for potential cell type-specific surface markers for human endoderm progenitors and leukemias, respectively. Although these platforms are very efficient for probing cell surface markers, they do not reveal association of specific markers with a particular function.
Beta cell specific surface markers may facilitate identification and characterization of embryonic beta cell progenitors as well as purification of homogeneous insulin-producing cells from cultures derived from hESCs or re-differentiated beta cells. They may also allow isolation of adult beta cells, thus contributing to future diagnostic applications. One cell surface marker which might be selective for human beta cells is TMEM27. While antibodies were recently raised against this antigen (Vats et al. 2012, Diabetologia 55(9): 2407-2416), it is still unclear if they can be used to purify beta cells by flow cytometry or other methods.
A variety of techniques have been previously employed in an attempt to isolate pancreatic beta cells. These include: genetic labeling (Meyer et al., Diabetes. 1998, 47, 1974-1977), Newport green dye labeling (Lukowiak B et al., J Histochem Cytochem. 2001, 49, 519-528), elimination of duct cells (Banerjee and Otonkoski 2009, Diabetologia 52(4): 621-625), and the generation of hybridoma-derived antibodies which can enrich for different endocrine and non-endocrine cell types (Dorrell et al., Stem Cell Res. 2008, 1, 183-194). None of these techniques, however, relies on beta cell-specific surface markers and the isolated cell populations still exhibit an unknown degree of heterogeneity. The same lack of marker information applies to other endocrine subsets in human pancreas (i.e. alpha cells, delta cells etc.).
There is an unmet need for robust screening procedures capable of identifying markers designating cells of desired type or function. Since tissue samples are often limited in quantity and availability, such procedures should permit functional analysis of multiple markers in parallel using small numbers of cells. Specifically, there is an unmet need for isolating insulin-producing beta cells within pancreatic tissue, for diagnostic and therapeutic uses in diabetes and other pancreatic-related disorders.